1. Field of the Invention
The invention relates generally to newly identified and isolated polynucleotides, proteins encoded by the polynucleotides, methods for producing proteins, and uses for the polynucleotides and proteins. More specifically, the current invention relates to bifunctional wax ester synthase proteins, polynucleotides encoding the proteins, and methods and compositions related thereto.
2. Description of Related Art
The capability for biosynthesis of neutral lipids is widely distributed in nature and is found in animals, plants as well as microorganisms. In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids serving as intracellular carbon and energy storage (Steinbüchel, 1991), but also few examples of substantial triacylglycerol (TAG) accumulation have been reported for species mainly belonging to the actinomycetes genera Mycobacterium (Barksdale and Kim, 1977), Nocardia and Rhodococcus (Alvarez et al., 1997) and Streptomyces (Olukoshi and Packter, 1994). Furthermore, biosynthesis of wax esters (oxoesters of long-chain primary fatty alcohols and long-chain fatty acids) has been frequently reported for members of the genus Acinetobacter (Fixter et al., 1986).
TAGs are the dominating storage lipid in animals, plants and eukaryotic microorganisms. TAG biosynthesis is involved in animals in numerous processes such as regulation of plasma TAG concentration, fat storage in adipocytes and milk production (Bell and Coleman, 1980). In plants, TAG synthesis is mainly important for the generation of seed oils (Lassner, 1997) Using diacylglycerol (DAG) as a substrate, three different classes of enzymes are known mediating TAG formation (Lehner and Kuksis, 1996). Acyl-CoA:DAG acyltransferase (DGAT) catalyzes the acylation of DAG using acyl-CoA as a substrate. Two DGAT families designated as DGAT1 and DGAT2 are known, which exhibit no sequence homologies to each other. Members of the DGAT1 gene family occur in animals and plants (Cases et al., 1998; Hobbs et al., 1999; Routaboul et al., 1999; Zou et al., 1999), whereas members of the DGAT2 gene family were found in animals (Cases et al., 2001), plants (Bouvier-Navée et al., 2000) and yeasts (Oelkers et al., 2002). In human, one DGAT1 related gene and five DGAT2 related genes were identified (Cases et al., 2001).
Recently, DGAT has attracted great interest since it is a potential therapeutical target for obesity treatment (Chen and Farese Jr., 2000). Acyl-CoA-independent TAG synthesis is mediated by a phospholipid:DAG acyltransferase found in yeast and plants, which uses phospolipids as acyl donors for DAG esterification (Dahlqvist et al., 2000). A third alternative mechanism present in animals and plants is TAG synthesis by a DAG-DAG-transacylase which uses DAG as acyl donor and acceptor yielding TAG and monoacylglycerol (Lehner and Kuksis, 1993; Stobart et al., 1997), but no gene coding such a transacylase could be identified yet.
Linear wax esters are lipophilic compounds containing a long chain fatty alcohol esterified to a long chain fatty acid. These wax esters are found in a number of diverse organisms ranging from mammals to plants to bacteria. For instance, wax esters are the principal component of spermaceti oil which, until recently, was obtained from the head cavity of sperm whales. Since the world-wide ban on whale hunting, however, the only natural source of wax esters on a commercial scale has been the seeds of jojoba, a bush or shrub that is adapted to growth in hot arid habitats. In jojoba plants, waxes are stored in the seeds of the plant where they serve as a means of energy storage for developing seedlings. Wax esters have also been found in several species of bacteria such as Acinetobacter calcoaceticus, a gram negative aerobic bacteria that accumulates wax esters when grown under nitrogen limited conditions. Wax esters from these bacterial sources, however, have not been utilized on a commercial scale.
Wax esters have a multitude of important commercial applications in a variety of technical areas, including the medical, cosmetics and food industries as well as their more traditional usage as lubricants for mechanical parts and the like. The wax esters obtained from jojoba can replace sperm whale oil in most or all traditional uses. They are useful for applications in cosmetics, as a lubricant, as an additive for leather processing, as a carrier for pharmaceuticals and as a solvent. Hydrogenation of the wax to eliminate double bonds produces a hard wax which is useful for surface treatments, in textile sizing, in coating paper containers and in cosmetics (e.g., lipstick and creams). Sulphurization of the wax or other modifications make the substance useful in specialty lubricant applications, as a textile softener, as a component of printing inks, and as a component in many technical products such as corrosion inhibitors, surfactants, detergents, disinfectants, plasticizers, resins and emulsifiers. For some of these applications the fatty alcohol derived by hydrolysis of the wax ester is the most valuable ingredient derived from the wax ester.
Because the yield of the jojoba plant is extremely low, however, the oil is relatively expensive compared with edible oils from plants or technically comparable materials from petroleum and its use has been limited to cosmetic products. Thus, a need exists to develop an alternate biological source of wax esters. One possibility, in this respect, is to recombinantly engineer a microbial species for efficient production of wax esters. Toward that end, information concerning enzymes and enzymatic pathways which are involved in wax ester biosynthesis, and the nucleic acid sequences that encode these enzymes are needed.
The most detailed information concerning wax ester biosynthesis concerns wax biosynthesis in jojoba plants, where it appears that two enzymes catalyze the formation of wax esters. The first step of the pathway is catalyzed by a fatty acyl-CoA reductase which reduces very long chain fatty acyl CoA (a very long chain fatty acyl CoA generally having greater than 18 carbons), and is known to catalyze the formation of a long chain alcohol directly from this substrate via an aldehyde intermediate. The second enzyme (wax ester synthase), an acyl-CoA-fatty alcohol transferase catalyzes the formation of an ester linkage between acyl-CoA and a fatty alcohol to yield a wax ester.
The pathway of wax ester biosynthesis in A. calcoaceticus, in contrast to the jojoba plant, comprises three enzymatic steps involved in the conversion of long-chain acyl-CoA to wax esters. In the first step, acyl-CoA is reduced by an NADPH-dependent acyl-CoA reductase to the corresponding fatty aldehyde. In the second step, the aldehyde is further reduced to the corresponding fatty alcohol catalyzed by the fatty aldehyde reductase. Finally, an acyl-CoA:fatty alcohol acyl transferase (wax ester synthase) condenses the fatty alcohol with acyl-CoA resulting in the formation of the wax ester.
Wax ester synthesis and WS activity have been reported for M. tuberculosis (Wang et al., 1972), and TAG accumulation and DGAT activity have been shown for M. smegmatis (Nakagawa et al., 1976; Wun et al., 1977). However, no proteins or genes have been reported to which these activities could be attributed.
Irrespective of the species involved, therefore, a key enzymatic step involved in wax ester biosynthesis is the transfer of an acyl chain from fatty acyl-CoA to a fatty alcohol, and this reaction is catalyzed by wax ester synthase. While several wax ester synthases have been described in terms of their substrate specificities and intracellular locations, very little is known about the proteins associated with this activity and the genes encoding this enzyme. In fact, the only gene encoding a wax ester synthase that has been identified is from jojoba. Thus, a need exists to identify genes encoding wax ester synthases from other species. In particular, a need exists to identify genes encoding wax ester synthases from a species that could be engineered to produce wax esters in large quantities and at a relatively affordable cost. The present invention addresses this need by providing polynucleotide sequences encoding bacterial wax ester synthases.